In Situ Casting of Platelet Rich Plasma/SiO2/Alginate for Bone Tissue Engineering Application in Rabbit Mandible Defect Model

Statement of the Problem: The administration of both platelet rich plasma (PRP) and silicon dioxide (SiO2) to the bone defects accelerates bone repair and regeneration. Appli-cation of both of them may show synergistic regenerative effects Purpose: Our objective was to evaluate the possible synergistic osteogenic effects of PRP and SiO2 by injecting them using an ad hoc device. Materials and Method: In this experimental study, PRP/SiO2 scaffolds were fabricated by in situ casting method with the help of CaCl2 as the gelation factor and alginate as the stroma; and then, the biodegradability and spatial arrangement were assessed. The injecta-ble scaffold was introduced into the 40 rabbit mandibular defects by an ad hoc two-channel injecting device. Five defects received PRP/SiO2/alginate as the treatment; the other sets of defects were treated by PRP/alginate, SiO2/alginate, and the last five defects served as the control groups by getting only alginate injections. The osteogenicity of the scaffolds was evaluated by radiological and histological procedures; they were then compared with each other. Analysis of variance and least significant difference tests were used to analyze the data Results: The SiO2-treated group showed a significant higher bone area compared to PRP/ SiO2-treated groups on day 40 (p= 0.013). The number of osteocytes was higher in SiO2-treated than the control groups on both 20 and 40 days (p= 0.032 and 0.022, respectively). The number of osteoclast was also higher in SiO2-treated than PRP-treated group (p= 0.028). In addition, the cells of this group had just started to create Haversian systems in newly formed bone tissues. Conclusion: Silica demonstrated a superior osteogenic activity over PRP in both short and long term periods. Evidently, they showed no synergistic regenerative effects. Our ad hoc device was efficiently capable of inserting the scaffolds into the injured sites with no diffi-culties or complications.


Introduction
The incidence of skeletal defects due to inactivity and obesity, particularly in societies with old population and advanced bone degenerative diseases, has dramatically increased and is expected to double this year [1]. In addition, the worldwide rate of accidental bone injury had a steeply upward trend over the past few years [2] and yet it remains a major challenge in the field of orthopedic surgery. Functional defects of the skeletal system usually happen as a result of trauma, injuries and diseases that can cause considerable complications and also various social and economic predicaments [3].
Hence, bone disorders extremely affect the patient's quality of life [4]. Trauma, cancer, and tuberculosis are the most common problems among the etiologies causing bone defects [5]. Particularly, mandibular defects are of utmost importance owing to the increasing prevalence and their effect on the matter of facial beauty and elegance. They are usually caused by trauma, removal of mandibular tumors, infection, and congenital diseases [6]. Surgery and bone grafting are the possible options for treatment of bone defects [5]. However, autogenous bone grafting is the most common measure to tackle these problems. It is known as the gold standard option due to its remarkable properties such as osteoconduction, osteoinduction, and osteointegrity [7]. Although autogenous bone grafting is the gold standard modality in treatment of skeletal and specifically mandibular defects, it results in several complications [8][9]. Nowadays, tissue engineering serves as an ideal alternative in dealing with skeletal defects [1]. Bioactive tissue engineered scaffolds enhance the cell differentiation, proliferation, migration, and angiogenesis, thereby improving ossification and bone formation [10].
One of recently innovative methods in bone tissue engineering is in situ casting of fluid biomaterials in the injured tissue and letting it solidify in the shape of the defect; therefore, we designed an ad hoc device to load biomaterials into the injured site. By using this device, not only is the treating procedure carried out much faster, but also the injected scaffold and the injured site are thoroughly superimposed. Moreover, it is very simple to use this device, and it does not require any specific prefabrication.
Natural biopolymers are widely utilized in this field due to their resemblance with extracellular matrix (ECM), convincing biologic function, and appropriate rate of biodegradability [11]. Recently, platelet rich plasma (PRP) has been useful in skeletal regenerative medicine due to its effect on accelerating the healing process [12][13][14]. Evidently, PRP is considered as a rich source of growth factors including platelet-derived growth factor (PDGF); transforming growth factor β (TGF-β); bone morphogenetic proteins (BMPs) as its subset; insulin-like growth factor (IGF-I); and vascular endothelial growth factor (VEGF) which are noticeably effective in angiogenesis, cell differentiation, proliferation and migration [14][15][16][17]. Its fibrin fibers form a biodegradable scaffold and are helpful in cell differentiation and proliferation [18]. Several studies with positive therapeutic results have been conducted using a PRP-based scaffold. For example, using a PRP and hydroxyapatite scaffold led to enhancement of ossification in lumbar vertebrae of rats [19]. On the other hand, nanoscale bioceramics have proved helpful to enhance cell adhesion, proliferation, and mechanical strength of the scaffold, thereby improving new integrated bone formation [11]. That is why they are frequently used in bone tissue engineering. They include hydroxyapatite, silicon dioxide (SiO 2 ), zirconium dioxide (ZrO 2 ), calcium phosphate (Ca 3 (PO 4 ) 2 ), calcium sulfate (CaSO 4 ), and so forth [20][21][22][23][24]. Among these materials, SiO 2 has drawn the attentions because of enhancing cell adhesions and improving cell viability and proliferation, which are essential for scaffold formation and ossification process [25][26]. According to in vivo studies, SiO 2 , also known as silica, increases proliferation of the endothelial cells and postoperative angiogenesis by accelerating production of VEGF, which plays a significant role in scaffold fabrication and bone formation [27]. Besides, Silica is involved in accelerating differentiation of the osteoblasts from the osteoprogenitor cells [28][29][30][31].
Likewise, subcutaneous transplantation of a combination of nanoporous silica, PRP and type I collagen stimulated the angiogenesis, mineralization and osteogenesis [23].
Nowadays, researchers prefer to choose the composites of biopolymers and bioceramics as bone tissue engineering material since they present benefits of both groups together in a single scaffold [11]. Several studies are in favor of their synergistic therapeutic effects [20,22,[31][32]. A combination of collagen, chitosan, and nanoparticles of hydroxyapatite resulted in bone tissue formation with high mechanical strength by increasing the differentiation, proliferation, and adhesion of cells [20]. A combination of hydroxyapatite and alginate were also used to deliver drugs for boosting osteoblast functions [31]. In addition, a composite of PRP, hydrox-yapatite and zirconia accelerated the osteogenesis and enhanced number of osteoblasts and osteocytes [22]. Therefore, we decided to combine PRP, SiO 2 and alginate and create a composite of biomaterials. The purpose of this study was to evaluate the possible synergic regenerative effects of PRP and silica-alginate injected by ad hoc device in rabbit mandible defect models.

Materials and Method
Scaffold fabrication PRP bank consisted of four 50 mL bags of human platelet serum provided from Fars blood transfusion center (Ghasrodasht Avenue, Shiraz, Fars province, Iran).
Heparin was added as an anticoagulant and the number of platelets was estimated to be1.042 ×10 6 /mL. They were aliquoted and frozen for less than 6 months and then thawed to be used in the structure of the scaffold.

Biodegradability test
Control, PRP, SiO 2, and PRP/SiO 2 scaffolds were fabricated in the culture dishes. Calcium chloride 2.5% was added to the scaffolds for electrogelation. Then, they were incubated at 37°C and 5% CO 2 for 20 min, so that the hydrogel scaffolds formed firmly. Thereafter, 0.01% trypsin enzyme (Sigma) was added to them, they were incubated for 12 hours, and then their weight was measured. The same procedure was done for the next 24, 48, 72, and 96 hours.

Experimental design
There were 4groups in both 20and 40 treatment periods (n=5). The defects in the group 1, also known as the control group, were filled with alginate, while the defects in groups 2, 3 and 4 were loaded with alginate/ SiO 2 , alginate/PRP and alginate/SiO 2 +PRP, respectively. After 20 and 40 days of the follow up, the rabbits were sacrificed and their mandibles were removed. The operated rabbits became conscious 1 hour after the surgery. They were transported in separate cages for being under control for 20 and 40 days; they had free access to food and water. They received daily intramuscular injection of penicillin/streptomycin for the first three postoperative days ( Figure 1).

Histological assessments
Operated rabbits were sacrificed on day 20 and 40 [33] according to the defined treatment planning, their mandibles were resected without muscles and fascia. Then,

Statistical tests
The data were analyzed using Analysis of variance and least significant difference tests and the p Value less than 0.05 was considered statistically significant. The graphs were depicted and the data analyzed by graph Pad6.  In addition, the cells cultured on PRP/SiO 2 scaffolds had the same appearance. Moreover, EDS test revealed that the percent of weight of SiO 2 in PRP/SiO 2 groups was 1.44 ± 0.8.

Biodegradability test
The data from biodegradation test revealed that at the first hours of incubation, the presence of PRP decelerated biodegradation, while as the time progressed; degradation rate was accelerated by incorporating PRP into the scaffolds (Figure 3).

Discussion
In the present study, we designed an injection device to introduce biomaterials for bone repair. This ad hoc devi- The results of the current study showed that PRP incorporation in the scaffolds led to decelerated degradation rate in short time, whereas, at long term, biodegradation was accelerated. It has been previously reported that platelet-rich fibrin degraded rapidly [36]. PRP incorporation in the scaffolds led to a decrease in the alginate concentration and it may be responsible for the high rate of degradation. On the other hand, it has been shown that the degradation rate of PRP scaffolds is related to the CaCl 2 concentration. As the CaCl 2 concentration increases, the degradation rate decreases [37].
We used 2.5% CaCl 2 that led to rapid disintegration of PRP-containing scaffolds. However, in short time, the combination of PRP and alginate decelerated the scaffold disintegration.
In the current study, we observed more bone regeneration and less connective tissue and osteoblasts per µm 2 on day 40 compared to day 20. The SiO 2 treated group had the most regenerated bone area and osteocytes per µm 2 on both days 20 and 40. Therefore, SiO 2 nanoparticles presented themselves as an agent for bone regeneration. Several similar studies have been conducted using the aforementioned agents [38][39][40]. Biosilica, as a biocompatible, inorganic polymer, has been shown to induce bone formation through enhancing mineralization [38], angiogenesis [39] and regulating immunoreactions [40]. The current work also confirms the positive influence of SiO 2 on accelerating the bone regeneration.
Combination of the other bioceramic such as HA with organic biomaterials has been shown synergistic impact on bone regeneration [19]. For instance, a com- Biphasic mineralized collagen scaffold containing intrafibrillar silica and apatite provoked the mouse mesenchymal stem cells to initiate osteogenesis [29]. Silicate composite with Graphene/polycaprolactone has been reported to provide a good osteoconductive scaffold for bone regeneration [41]. SiO 2 /PRP/bone substitute biomaterial has been suggested for replacing the bone, and it was found that SiO 2 influences in vitro releasing pattern of growth factors by platelet population [42]. Our previous in vitro study also revealed a composite of SiO 2 and PRP has led to appropriate osteoblasts viability, proliferation, and function [43]. In contrast to the previous in vitro investigations, the current study did not indicate synergistic impact on bone regeneration potential by implanting the SiO 2 /PRP/alginate scaffold.
Another study was performed utilizing a composite of PRP, mesenchymal stem cells, and nanoporous silicon enclosures as an osteogenic scaffold implanted subcutaneously, which resulted in bone formation and angiogenesis [23]. However, there are some studies that reject the bone-regenerating characteristics of PRP [22,[44][45][46]. For instance, PRP failed to promote bone reconstruction in a canine defect model. In fact, it presented lower amounts of bone formation than the non-PRP group [47]. In the present study, we observed the same behavior from PRP when it was simultaneously used with silica. Not only it did not enhance the bone reconstruction in the injured site, but it also suppressed the osteogenic activity of silica. Consequently, they do not  tive potential of all the scaffolds was the same [22]. We chose to evaluate the osteogenic capacity of the scaffolds 20 days after the surgery and we believe that short term assessments may need to find accelerating potential of such combination.
Our study showed that the combination of SiO 2 and PRP had no impact on the number of osteoclasts, while in short time, the number of osteoclast increased by SiO 2 administration. Mesenchymal stem cell differentia-tion potential into osteoclasts was evaluated on SiO 2 in combination with CaO scaffolds, and it was found that the differentiation, survival, and adherence of osteoclast precursors were influenced by culturing on the scaffold [48]. Besides, adding SiO 2 to poly(lactic-co-glycolic)acid membrane increases the number of osteoclast in rabbit calvaria defect model [49]. In another study, the osteoclastogenesis ability of SiO 2 /collagen was compared with hydroxyapatite. Silicone-containing scaffolds increased the bone resorption compared to hydroxy-apatite-containing scaffolds [50]. On the other hand, PRP has been recorded to inhibit [51][52] or stimulate [53] the osteoclast differentiation through various mechanisms based on the preparation procedure. In the current study, the number of osteoclasts was similar in the PRP/SiO 2 -, PRP-treated and control animals that may be attributed to the contradictory effects of PRP andSiO 2 on osteoclastogenesis as well as the way of PRP and SiO 2 preparation.
The current study had several limitations. Firstly, it was better to sacrifice the rabbits and obtain the samples

Conclusion
That the results of the current study showed that osteogenesis was superior in SiO 2 -treated defects compared to the other groups. The combination of PRP and SiO 2 did not show any synergistic influence on bone regeneration. Besides, the injectable scaffold could be introduced into the defect by ad hoc device without any adverse impact.